Clostridium perfringens bacteriophage and uses thereof

ABSTRACT

The present invention is directed to isolated bacteriophage having strong lytic activity against strains of  Clostridium perfringens , and methods of using that bacteriophage, and/or progeny or derivatives derived therefrom, to control the growth of  Clostridium perfringens  in various settings.

FIELD OF THE INVENTION

The present invention relates to novel bacteriophages designated CPAS-7,CPAS-12, and CPAS-15 (the “Deposited bacteriophage”), and compositionsand preparations corresponding thereto. More specifically, isolatedbacteriophage preparations possessing lytic activity against strains ofClostridium perfringens (the “Targeted Bacteria”) are provided in orderto control the growth of the Targeted Bacteria, which will reduce theirability to contaminate and colonize various environments, including butnot limited to (i) raw, unprocessed food products, (ii) equipment usedto process or manufacture various food products, (iii) various foodproducts processed or manufactured with equipment contaminated with theTargeted Bacteria, (iv) animals contaminated with the Targeted Bacteria,and (v) animal environments contaminated with the Targeted Bacteria. Theinvention also provides methods for detecting the presence of theTargeted Bacteria in processed or unprocessed food products, and inequipment used to process or manufacture the food products. In addition,the invention provides methods of using the Deposited bacteriophage toremove the Targeted Bacteria from medical, veterinary, animal husbandry,and other environments where they may be passed to humans or animals.Also, the invention additionally provides methods of using thebacteriophage to prevent and treat animal and human diseases caused bythe Targeted Bacteria.

BACKGROUND OF THE INVENTION

Bacteriophages are bacterial viruses that attach to their specific hostsand kill them by internal replication and bacterial lysis involving acomplex lytic cycle involving several structural and regulatory genes.Phages are very specific in that they only attack their targetedbacterial hosts. They cannot infect human or other eukaryotic cells.Bacteriophages were first identified, in the early part of the 20thcentury by Frederick Twort and Felix D'Herelle who called thembacteriophages or bacteria-eaters (from the Greek phago meaning to eator devour) (Duckworth, D. H. (1976). Who discovered bacteriophage?Bacteriol Rev 40(4): 793-802; Summers, W. C. (1999). Bacteriophagediscovered. Felix d'Herelle and the origins of molecular biology. NewHaven, Conn., Yale University Press: 47-59). At that time, with the ageof antibiotics still in the future, bacteriophages were considered to bea potentially powerful cure for bacterial infections, and they weretherapeutically utilized throughout the world during the pre-antibioticera. The use of phages in humans was found to be very safe; however,phage therapy did not always work and, with the advent of antibioticsthat were effective against a broad spectrum of pathogenic bacteria, itgradually fell out of favor in the United States and Western Europe.Several factors (reviewed in more detail in Sulakvelidze, A., Z.Alavidze, et al. (2001). Bacteriophage therapy. Antimicrob AgentsChemother 45(3): 649-659; Summers, W. C. (2001). Bacteriophage therapy.Ann Rev Microbiol 55: 437-51), including the lack of a broadunderstanding of phage biology and inadequate diagnostic bacteriologytechniques, contributed to the failure of some of the early phagetherapy studies and to the associated decline of interest in phagetherapy in the West. At the same time, phage therapy continued to beutilized in the former Soviet Union and Eastern Europe, where phagetherapy still is being used to treat a wide range of bacterial diseasesranging from intestinal infections to septicemia. Comprehensiveinformation about human and veterinary applications of bacteriophageshas been recently reviewed by several investigators (Alisky, J., K.Iczkowski, et al. (1998). Bacteriophages show promise as antimicrobialagents. J Infect 36(1): 5-15; Summers, W. C. (2001). Bacteriophagetherapy. Annu Rev Microbiol 55: 437-51; Merril, C. R., D. Scholl, et al.(2003). “The prospect for bacteriophage therapy in Western medicine.”Nat Rev Drug Discov 2(6): 489-497; Sulakvelidze, A. and P. Barrow(2005). Phage therapy in animals and agribusiness. Bacteriophages:Biology and Applications. E. Kutter and A. Sulakvelidze. Boca Raton,Fla., CRC Press: 335-380; Sulakvelidze, A. and E. Kutter (2005).Bacteriophage therapy in humans. Bacteriophages: Biology andApplication. E. Kutter and A. Sulakvelidze. Boca Raton, Fla., CRC Press:381-436).

Despite the use of bacteriophage in various practical settings,including the treatment of diseases in various animals, there remains inthe art a need for the discovery of novel bacteriophages, selection ofoptimal bacteriophages for specific practical applications, andidentifying methods for using these bacteriophages in several criticalareas, including clinical applications, food safety-related uses andenvironmental decontamination. For example, one significant needconcerns the treatment of processed or unprocessed food products toreduce, eliminate or prevent colonization with undesirable bacteria suchas pathogens responsible for food-borne illness and food spoilageorganisms. A second critical area of need concerns the removal ofundesirable bacteria from industrial environments such as foodprocessing facilities to prevent colonization thereof. A third criticalarea of need concerns the removal of antibiotic resistant organisms fromenvironments where they may be passed to susceptible humans and animals,such as hospitals, nursing homes, veterinary facilities, and other suchenvironments. Additionally, new bacteriophage and methods of using thesame are needed for the prevention or treatment of animal and humanbacterial disease, particularly those diseases caused byantibiotic-resistant organisms.

SUMMARY OF THE INVENTION

The invention meets the described needs and more by providingcompositions comprising novel CPAS-7, CPAS-12, and CPAS-15 bacteriophagehaving lytic specificity for the Targeted Bacteria. The inventionadditionally provides methods of using the Deposited bacteriophage tocontrol or prevent the infection or colonization of processed andunprocessed food products by Targeted Bacteria, or colonization ofequipment involved in the processing of the same food product(s). Theinvention additionally provides methods of using the Depositedbacteriophage to prevent, eradicate, or reduce the levels ofcolonization of various animals (including humans) with TargetedBacteria. The invention also provides methods of detecting the presenceof Targeted Bacteria cells on processed or unprocessed food products, orequipment involved in the processing of the same food products. Theinvention additionally provides methods of using the Depositedbacteriophage for the removal of antibiotic-resistant or otherundesirable pathogens from medical, veterinary, animal husbandry, andother environments where they may be passed to humans or animals. Theinvention additionally provides for methods of using the Depositedbacteriophage to prevent or treat human and/or other animal diseasescaused by Targeted Bacteria

BRIEF DESCRIPTION OF THE FIGURES

Figures

FIG. 1 shows a Restriction Fragment Length Polymorphism (RFLP) profileof the CPAS-7, CPAS-12, and CPAS-15 bacteriophage in comparison to twoother bacteriophages also specific for the Targeted Bacteria. FIG. 1.The figure shows an XmnI digest of bacteriophage DNA following agarosegel electrophoresis. Note the unique patterns of the DepositedBacteriophage, CPAS-7, CPAS-12, and CPAS-15. Lane M, GeneRuler DNALadder Mix (Fermentas); Lane 1, CPLV-42; Lane 2, CPAS-7; Lane 3,CPAS-15; Lane 4, CPTA-37; Lane 5, CPAS-12.

Tables

Table 1 shows the lytic specificity of the Deposited Bacteriophage forClostridium perfringens, the Targeted Bacteria.

TABLE 1 C. perfringens Deposited Bacteriophage Host Strain CPAS-12CPAS-15 CPAS-7 ATCC 13124 + + + Cp 1 − − − Cp 2 + + + Cp 3 + − − Cp4 + + − Cp 5 + + − Cp 6 + − − Cp 7 + + + Cp 8 − + + Cp 9 + + − Cp 10 − +− Cp 11 + − − Cp 12 + − − Cp 13 + + − Cp 14 − + + Cp 15 + − − Cp16 + + + Cp 17 + + + Cp 19 + − − Cp 21 + − − Cp 22 + + − Cp 23 + + + Cp24 + + − Cp 25 + + + Cp 26 + − − Cp 27 + − − Cp 28 − − − Cp 29 − − + Cp30 + − − Cp 31 + − − Cp 32 − + + Cp 33 + + + Cp 34 − + + Cp 35 − + + Cp36 + + − Cp 37 + − − Cp 38 + + + Cp 39 − − − Cp 40 − − − Cp 41 + − + Cp42 + + + Cp 43 + − − Cp 44 + − + Cp 45 + − − Cp 46 − − − Cp 47 + + +

Table 2 shows the lytic specificity of the Deposited Bacteriophage fornon-targeted bacteria of differing bacterial species.

TABLE 2 Deposited Bacteriophage Non-C. perfringens Strain CPAS-12CPAS-15 CPAS-7 Pseudomonas aeruginosa Pa 1 − − − Pa 3 − − − Pa 7 − − −Pa 15 − − − Pa 21 − − − Pa 33 − − − Pa 42 − − − Pa 62 − − − Pa 65 − − −Pa 72 − − − Salmonella enteric SE 24 − − − SS 28 − − − ST 31 − − − SHE43 − − − SH 49 − − − S 45 − − − S AE 72 − − − SK 103 − − − SR 114 − − −SH 162 − − − Listeria monocytogenes Lm 6 − − − Lm 10 − − − Lm 23 − − −Lm 31 − − − Lm 35 − − − Lm 49 − − − Lm 62 − − − Lm 67 − − − Lm 79 − − −Lm 86 − − − Escherichia coli Ec 3 − − − Ec 26 − − − Ec 37 − − − Ec 41 −− − Ec 56 − − − Ec 60 − − − Ec 65 − − − Ec 68 − − − Ec 73 − − − Ec 77 −− −

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions

As used herein, “isolated” will mean material removed from its originalenvironment in which it naturally occurs, and thus is altered by thehand of man from its natural environment. Isolated material may be, forexample, foreign nucleic acid included in a vector system, foreignnucleic acid contained within a host cell, or any material which hasbeen removed from its original environment and thus altered by the handof man. Isolated material further encompasses bacteriophage specific forthe Targeted Bacteria or particular Targeted Bacteria isolates, isolatedand cultured separately from the environment in which it was located,where these isolates are present in purified compositions that do notcontain any significant amount of other bacteriophage or bacterialstrains, respectively.

As used therein, “Deposited bacteriophage” will mean isolatedbacteriophage CPAS-7, CPAS-12, and CPAS-15 deposited with the ATCC onJun. 15, 2007, and receiving ATCC Deposit Accession No. PTA-8478 forCPAS-7, PTA-8479 for CPAS-12, and PTA-8480 for CPAS-15.

As used therein, “Targeted Bacteria” will mean Clostridium perfringens.

As used herein, “progeny” shall mean replicates of the Depositedbacteriophage, including descendants of the Deposited bacteriophagecreated by serial passage of the Deposited bacteriophage or by othermeans well known in the art, or bacteriophage whose RFLP profiles aresubstantially equivalent to the RFLP profile of the Depositedbacteriophage of FIG. 1. The term substantially equivalent is used todescribe variability between organisms in accordance with the standardsadvanced by Tenover et al. from the United States Centers for DiseaseControl and Prevention (Tenover, F. C. et al. (1995) InterpretingChromosomal DNA Restriction Patterns Produced by Pulsed-Field GelElectrophoresis: Criteria for Bacterial Strain Typing. J. Clin.Microbiol. 33:2233-2239). Tenover et al. teaches the acceptable levelsof variation that may be seen when the genomes of identical propagatedorganisms are electrophoretically analyzed following restriction enzymedigestion.

As used herein, “recombinant bacteriophage” shall mean all geneticallymodified versions of the Deposited bacteriophage or its progeny,obtained by serial passaging (in vivo or in vitro) or geneticmanipulations of the Deposited bacteriophage or its progeny. Suchmanipulations include, but are not limited to, introducing genes or genecassettes encoding alternative proteins or nonfunctional proteins, ornoncoding nucleotide sequences into the genome of the Depositedbacteriophage.

As used herein, “derivatives” shall mean all substances that constitutesubunits or expression products of the Deposited bacteriophage or itsprogeny, including (but not limited to) phage nucleic acids, partial orcomplete phage genes, gene expression products, and structuralcomponents. For example, derivatives of the invention meanpolyribonucleotide(s) and polydeoxyribonucleotide(s), including modifiedor unmodified bacteriophage DNA, cDNA, mRNA and synthetic polynucleotidesequences, as well as DNA/RNA hybrids. Polynucleotides of the inventionalso encompass modified polynucleotides, such as for examplephosphorylated DNAs.

As used herein, “substantially pure” will mean material essentially freeof any similar macromolecules that would normally be found with it innature. For example, a substantially pure bacteriophage is in acomposition that contains no more than 1% of other bacteriophages.

As used herein, “bacteriophage composition” will mean a compositioncomprising, or alternatively consisting essentially of, or alternativelyconsisting of, the Deposited bacteriophage. A “bacteriophagecomposition” as used herein does not include the Deposited bacteriophageas it exists in its natural environment prior to isolation and/orsubstantial purification.

As used herein, “colonization” or “colonized” will refer to the presenceof Targeted Bacteria on foodstuff(s), or environmental surface(s), or invivo such as in the gastrointestinal tract or skin of a mammalianorganism without perceptible significant alteration other than thepresence of bacteria. The terms “colonization” and “colonized” stand incontrast to the terms “infection” or “infected” which are commonlyunderstood to require perceptible deleterious alteration as part oftheir definition. “Colonization” and “colonized” may also refer to thepresence of bacteria in or on a human or animal without perceptibledamage, alteration, or disease.

As used herein, “ATCC” will mean the American Type Culture Collection,located at 10801 University Boulevard, Manassas, Va., 20110-2209, USA.

As used herein, “ORF” will mean an Open Reading Frame which is anin-frame sequence of codons that (in view of the genetic code)correspond to or encode a protein or peptide sequence. Two ORFscorrespond to each other if the sequences or their complementarysequences encode the same amino acid sequences. An ORF sequence,operably associated with appropriate regulatory sequences, may betranscribed and translated into a polypeptide in vivo. A polyadenylationsignal and transcription termination sequence will usually be located 3′to the coding sequence.

The Deposited Bacteriophage

The Deposited Bacteriophage has binding specificity for TargetedBacteria, and is capable of lysing Targeted Bacteria. The inventionfurther contemplates variants of the Deposited Bacteriophage, which arebacteriophage having minor variation(s) in the genomic sequence andpolypeptides encoded thereby while retaining the same general genotypicand phenotypic characteristics as the Deposited Bacteriophage. Suchvariants are considered to be the Deposited Bacteriophage in accordancewith the standards advanced by Tenover et al. from the United StatesCenters for Disease Control and Prevention (Tenover, F. C. et al. (1995)Interpreting Chromosomal DNA Restriction Patterns Produced byPulsed-Field Gel Electrophoresis: Criteria for Bacterial Strain Typing.J. Clin. Microbiol. 33:2233-2239). The invention also contemplatesprogeny and bacteriophage derivative(s).

The invention contemplates the use of the Deposited Bacteriophage, andits progeny and derivatives, to control the growth on, or colonizationof, processed and unprocessed food products by Targeted Bacteria, or thecolonization of buildings and equipment, particularly those associatedwith the processing of the same food product. The invention alsoprovides methods of identifying Targeted Bacteria as a bacterialdiagnostic and/or detecting the presence of Targeted Bacteria onprocessed or unprocessed food products, or equipment or buildings suchas those involved in the processing of the same food products. Theinvention further provides methods of using the Deposited Bacteriophagefor the removal of antibiotic-resistant or other undesirable pathogensfrom medical, veterinary, animal husbandry, or any additionalenvironments where they may be passed to humans or animals. Theinvention additionally provides for methods of using the DepositedBacteriophage to prevent and/or treat human and animal diseases causedby Targeted Bacteria. The Deposited Bacteriophage is administered forthe methods of the invention as a homogenous phage administration, oralternatively as a component of a multi-phage composition comprisingseveral bacteriophages. These methods of use are provided with greaterparticularity infra.

Use of the Deposited Bacteriophage and Its Progeny

Food Preservation

In one embodiment, the invention contemplates a method for theprevention of foodborne illnesses caused by the Targeted Bacteria,comprising contacting a food product or products with a microbial growthinhibiting effective amount of a bacteriophage composition comprisingthe Deposited Bacteriophage. The modes of contact include, but are notlimited to, spraying or misting the Deposited Bacteriophage compositionon the food product(s), or by dipping or soaking the food product(s) ina solution containing a concentration of the Deposited Bacteriophagesufficiently high to inhibit the growth of Targeted Bacteria, or adding,injecting or inserting the Deposited Bacteriophage into the foodproduct(s).

In another embodiment, the invention contemplates the application of theDeposited Bacteriophage composition to equipment associated with theprocessing of food product(s), such as cutting instruments, conveyorbelts, and any other implements utilized in the mass production of foodproducts, including the preparation, storage and packaging steps of foodprocessing. The Deposited Bacteriophage can additionally be introducedinto packaging materials used to contain food product(s), prior to orfollowing transfer of the food product(s) to the packaging materials.Alternatively the Deposited Bacteriophage can be useful in the localprocessing of food products located, for example, in the home or in arestaurant kitchen, using the same modes of contact as described supra.

In another embodiment of the invention, the Deposited Bacteriophage isadded as a component of paper products, either during processing orafter completion of processing of the paper products. Paper products towhich the Deposited Bacteriophage may be added include, but are notlimited to, paper towels, toilet paper, moist paper wipes. In apreferred embodiment of the invention, the Deposited Bacteriophage isadded as a component of cleansing wipes. The Deposited Bacteriophage maybe added in an aqueous state to a liquid-saturated paper product, oralternatively may be added in powder form, such as lyophilized, to drypaper products, or any combination thereof. In similar manner, theDeposited Bacteriophage may be incorporated into films such as thoseused for packaging foods, such as by impregnating or coating the film.

The methods of the invention further contemplate the application of theDeposited Bacteriophage to the floors, walls, ceilings, drains, or otherenvironmental surfaces in structures such as the industrial foodprocessing, military, or home environments. In a particularly preferredembodiment of the invention, the Deposited Bacteriophage is applied torefrigerated devices used to store or transport food or food products,including but not limited to, home and industrial refrigerators,deli-meat and cheese counters, refrigerated trucks, and mobilefood-service vehicles.

In a non-limiting embodiment of the invention, the DepositedBacteriophage of the invention is useful in preventing the colonizationof, or inhibiting the growth of, Targeted Bacteria on processed orunprocessed food products by infecting, lysing or inactivating TargetedBacteria present on said food product. Processed or unprocessed foodproducts in which the Deposited Bacteriophage is particularly useful inpreventing the growth or colonization of Targeted Bacteria include, butare not limited to beef (particularly ground beef), food products madewith ground beef such as hamburgers, sloppy joes, lasagna, stews, andother ground beef preparations, fresh vegetables exposed to TargetedBacteria presumably via animal waste, such as lettuce, spinach, greenonions, and other fresh vegetables commonly grown out of doors infields, drinking water, and foodstuffs secondarily contaminated withTargeted Bacteria through contact with contaminated foods, sewage, oranimal feces.

The Deposited Bacteriophage can also be administered to potable andnon-potable water sources to reduce or eliminate the presence ofTargeted Bacteria.

Bacteriophage compositions of the invention may be provided in aqueousor non-aqueous embodiments for the preservation of food.

Aqueous embodiments of the Deposited Bacteriophage include aqueouscompositions comprising, or alternatively consisting of, the DepositedBacteriophage alone or in combination with other bacteriophage orbacteriophages. Aqueous embodiments of the Deposited Bacteriophage areavailable in solutions that include, but are not limited to, phosphatebuffered saline, Luria-Bertani Broth or chlorine-free water.

Non-aqueous embodiments of the Deposited Bacteriophage include, but arenot limited to, lyophilized compositions or spray-dried compositionscomprising, or alternatively consisting of, the Deposited Bacteriophagealone or in combination with other bacteriophage(s). Freeze-dried andspray-dried compositions may also include soluble and/or insolublecarrier materials as, for example, processing aids.

The Deposited Bacteriophage can be administered at a concentrationeffective to prevent the initial colonization of foods with TargetedBacteria, or to inhibit the growth or colonization of food or foodproducts, as well as the equipment used to process or store food. In anon-limiting embodiment of the invention, the Deposited Bacteriophagestypically administered at a growth inhibiting effective amount of aconcentration of about 10⁷ to about 10¹¹ Plaque Forming Units (PFU)/ml.One of skill in the art is capable of ascertaining bacteriophageconcentrations using widely known bacteriophage assay techniques (Adams,M. H. (1959). Methods of study bacterial viruses. Bacteriophages.London, Interscience Publishers, Ltd.: 443-519.). The Depositedbacteriophage at such concentrations may be applied at, for example,about 1 ml/500 cm² of food surface.

Environmental Control

In another embodiment of the invention, the Deposited Bacteriophage isadministered to environments to control the growth or viability ofTargeted Bacteria. Environments in which the Deposited Bacteriophage isuseful to control the growth or viability of Targeted Bacteria include,but are not limited to, abattoirs, meat processing facilities, feedlots,vegetable processing facilities, medical facilities (includinghospitals, out-patient clinics, school and/or university infirmaries,and doctors offices), military facilities, veterinary offices, animalhusbandry facilities, public and private restrooms, and nursing andnursing home facilities. The invention further contemplates the use ofthe Deposited Bacteriophage for the battlefield decontamination of foodstuffs, the environment, and personnel and equipment, both military andnon-military.

The Deposited Bacteriophage is additionally useful alone or incombination with other bacteriophage(s) and/or other compounds, forpreventing the formation of biofilms, or controlling the growth ofbiofilms, in various environments. Aqueous embodiments of the DepositedBacteriophage are available in solutions that include, but are notlimited to, phosphate buffered saline, Luria-Bertani Broth orchlorine-free water. In a particularly preferred embodiment, theDeposited Bacteriophage is used to control biofilm formation and growthin municipal water systems, industrial water systems, and personal watersystems, as well as biofilms present in refrigerated environments.

The modes of administration include, but are not limited to, spraying,hosing, and any other reasonable means of dispersing aqueous ornon-aqueous Bacteriophage compositions, in an amount sufficiently highto inhibit the growth or viability of Targeted Bacteria. In anon-limiting embodiment of the invention, the Deposited Bacteriophage isuseful in preventing the growth or viability of Targeted Bacteria byinfecting, lysing or inactivating Targeted Bacteria present in saidenvironment. Administration of the Deposited Bacteriophage compositionincludes application to the floors, walls, counter-tops, ceilings,drains or any other environmental surface.

Bacteriophage compositions of the invention are available in aqueous ornon-aqueous embodiments discussed earlier for Food Preservationapplications.

In another embodiment of the invention, the Deposited Bacteriophage isadded as a component of paper products, either during processing orafter completion of processing of the paper products. Paper products towhich the Deposited Bacteriophage may be added include, but are notlimited to, paper towels, toilet paper, and moist paper wipes. In apreferred embodiment of the invention, the Deposited Bacteriophage isadded as a component of cleansing wipes; it may be added in an aqueousstate to a liquid-saturated paper product, or alternatively may be addedin powder form such as a lyophilized preparation, to dry paper products,or any combination thereof.

The Deposited Bacteriophage can be administered at a concentrationeffective to inhibit the growth or viability of Targeted Bacteria in aparticular environment. In a non-limiting embodiment of the invention,the Deposited Bacteriophage is administered at a concentration of about10⁷ to 10¹¹ PFU/ml. One of skill in the art is capable of ascertainingbacteriophage concentrations using widely known bacteriophage assaytechniques (Adams, M. H. (1959). Methods of study bacterial viruses.Bacteriophages. London, Interscience Publishers, Ltd.: 443-519.).

Prevention or Treatment of Infection or Colonization

In another embodiment, the invention contemplates a method for theprevention or treatment of illnesses caused by the Targeted Bacteria,comprising contacting a microbial growth inhibiting effective amount ofa bacteriophage composition comprising the Deposited Bacteriophage witha site or sites of infection of a host mammal infected with TargetedBacteria. For example, among several bacteria that cause significantmorbidity and mortality in chickens, C. perfringens is one of the mostnotorious pathogens. In chickens, C. perfringens infections are oftenmanifested as necrotic enteritis that occur later in the productioncycle, often following a coccidial infection or other insult to the GItract.

The infected mammalian host may be a human host or animal host. Inparticular, the host may be a bovine, poultry, or porcine host.Prevention of the infection by Targeted Bacteria, or treatment ofinfected persons or animals, is particularly preferred inimmuno-compromised persons, pregnant females, and newborns and infants,who maybe at an elevated risk of infection by Targeted Bacteria. Themodes of contact include, but are not limited to, spraying or mistingthe bacteriophage composition on the infected mammalian host, byinjecting at a site or sites of infection a pharmaceutically acceptablecomposition containing a concentration of the Deposited Bacteriophagesufficiently high to inhibit the growth of Targeted Bacteria, or byingesting a solution containing a concentration of the DepositedBacteriophage sufficiently high to inhibit the growth of TargetedBacteria. Additional routes of administration include but are notlimited to oral, rectal, topical, ophthalmic, buccal, intravenous, otic,nasal, vaginal, inhalation, and intrapleural.

In another nonlimiting embodiment of the invention, the DepositedBacteriophage is useful for preparing bacterial vaccines or bacterinsthat eliminate or reduce colonization of the Targeted Bacteria in,and/or their being shed by, various agriculturally-important animals.One example of a practical application for that type of vaccine is inthe cattle-raising industry, where its administration may significantlyreduce colonization of cattle with the Targeted Bacteria; thus,improving public safety by reducing contamination of beef with theTargeted Bacteria.

Bacteriophage compositions of the invention are available in aqueous ornon-aqueous embodiments discussed earlier for Food Preservationapplications.

The Deposited Bacteriophage can be administered at a concentrationeffective to inhibit the growth or viability of Targeted Bacteria in theinfected host. In a non-limiting embodiment of the invention, theDeposited Bacteriophage is administered at a concentration of about 10⁷to 10¹¹ PFU/ml. One of skill in the art is capable of ascertainingbacteriophage concentrations using widely known bacteriophage assaytechniques (Adams, M. H. (1959). Methods of study bacterial viruses.Bacteriophages. London, Interscience Publishers, Ltd.: 443-519.)

Depending on the severity of peculiarities of the infection, theDeposited Bacteriophage can be administered to animals (includinghumans) (i) orally, in tablet or liquid formulation (10⁵-10¹¹ PFU/dose),(ii) rectally, (iii) locally (skin, eye, ear, nasal mucosa, etc.), intampons, rinses and creams, (iv) as aerosols or intrapleunal injectionsand (v) intravenously.

Use of Recombinant Bacteriophage

In one embodiment of the invention, homologous recombination techniquesare used to introduce sequences encoding alternative proteins,non-functional proteins, or non-coding sequences into the bacteriophageDNA sequence. Such techniques are useful to “knock-out” undesired traitsof the Deposited Bacteriophage, or alternatively to introduce differenttraits. In a particularly preferred embodiment of the invention,homologous recombination is used to “knock-out” ORFs encoding proteinsthat maybe involved in a lysogenic cycle of the bacteriophage.

In another embodiment of the invention, homologous recombination is usedto introduce or knock-out genes involved in burst size. For example,homologous recombination is used to introduce alternative bacteriophagegenes which delay the burst event or increase the phage burst size.References disclosing alternative bacteriophage genes involved in thetiming of the burst event or the size of the phage burst include, butare not limited to (Johnson-Boaz, R., C. Y. Chang, et al. (1994). “Adominant mutation in the bacteriophage lambda S gene causes prematurelysis and an absolute defective plating phenotype.” Mol Microbiol 13(3):495-504; Wang, I. N., D. L. Smith, et al. (2000). “Holins: the proteinclocks of bacteriophage infections.” Annu Rev Microbiol 54: 799-825).

In another embodiment of the invention, recombinant bacteriophageharboring reporter system(s) is generated for various practicalapplications. One example of possible application of such system isspecies identification/confirmation of Targeted Bacteria as bacterialdiagnostics. Another possible application is the detection of thepresence of viable cells of Targeted Bacteria to which the Depositedbacteriophage have specificity. Following the techniques of Loessner etal., for example, one of skill in the art can generate recombinantreporter bacteriophage (Loessner, M. J., C. E. Rees, et al. (1996).“Construction of luciferase reporter bacteriophage A511::luxAB for rapidand sensitive detection of viable Listeria cells.” Appl EnvironMicrobiol 62(4): 1133-1140.). For example, the Vibrio harveyi luxAB genemay be introduced into the bacteriophage DNA sequence using techniquessuch as homologous recombination. An ideal target for the introductionof the luxAB gene is immediately downstream and in frame with an ORFencoding bacteriophage capsid protein, thereby creating a sequenceencoding a fusion protein. The preferable location of introduction ofthe luxAB gene sequence is particularly before any sequence encoding atranscriptional terminator downstream of the ORF encoding a capsidprotein. Other bacteriophage ORF sequences which may function as usefulsources of luxAB gene-fusions include gene sequences encodingtail-sheath proteins, or any other late gene region sequences encodingphage head or tail proteins. The resulting recombinant bacteriophage maybe used with methods of the invention to detect the presence of viablecells of Targeted Bacteria.

In addition to the Vibrio harveyi luxAB gene, other reporter genes whichare useful for the generation of reporter bacteriophage include, but arenot limited to, the firefly luciferase gene.

The invention further contemplates the introduction of one or more ofthe above-described recombinant events. For example, a recombinantbacteriophage of the invention may harbor one or more reporter gene(s)as well as lack one or more genes associated with certain undesirablebiological functions of the bacteriophage.

Use of Bacteriophage Derivatives

Derivatives, such as polypeptides, including but not limited tobacteriophage lytic enzymes, encoded by the bacteriophage or thebacteriophage progeny are used for applications designed to prevent thegrowth of Targeted Bacteria through cell wall lysis. In this context,lytic polypeptides are useful for the prevention of the growth ofTargeted Bacteria on processed and unprocessed food products, as well asequipment used for the processing of said food products.

In another preferred embodiment of the invention, bacteriophagederivatives are useful for the treatment of one or more infections in amammal, including humans, by administering their therapeuticallyeffective amounts to the patient. This method is useful for thetreatment of infections of the gastrointestinal system. Similarly, thismethod is useful in a prophylactic setting for the prevention ofinfection by Targeted Bacteria in pregnant mammals, including humans.This method of treatment is further useful for the prevention or otherdisorders or infections caused by Targeted Bacteria, such as acutebloody or non-bloody diarrhea, sometimes associated withhemolytic-uremic syndrome.

Another nonlimiting embodiment of the invention is that thebacteriophage derivatives such as lysins will be useful for preparingbacterial vaccines or bacterins that eliminate or reduce colonization ofthe Targeted Bacteria in, and/or their being shed by, variousagriculturally-important animals. One example of a practical applicationfor that type of vaccine is in the cattle-raising industry, whereadministration of such vaccines/bacterins may significantly reducecolonization of cattle with the Targeted Bacteria; thus, improvingpublic safety by reducing contamination of beef with the TargetedBacteria.

Detection Systems

The Deposited bacteriophage, its progeny, recombinant bacteriophage, orderivatives of the above are useful in methods of screeningenvironmental samples (including food products and food processingequipment) and clinical specimens for the presence of viable cells ofTargeted Bacteria. For example, in one such system, recombinantbacteriophage containing a reporter system such as, for example, aluciferase reporter system is applied to the sample and analyzed at sometime point in the future for the activation of the reporter molecule.The activation of the reporter molecule is indicative of the presence ofviable cells of Targeted Bacteria.

The Deposited bacteriophage, its progeny, recombinant bacteriophage, orderivatives such as lytic enzymes are useful in methods of screeningenvironmental samples including food products and food processingequipment and clinical specimens for the presence of viable cells ofTargeted Bacteria, by monitoring and measuring bacterial metabolismproducts such as bacterial adenosine kinase (AK) or adenosinetriphosphate (ATP) released as a result of specific lysis of TargetedBacteria. For example, when the released ATP is incubated with aluciferin/luciferase mixture, a rapid flash of peak light emissionoccurs within less than a second, followed by a steady glow lasting forseveral hours. By measuring the luminescence, it is possible to obtain aquantitative estimate of the number of bacterial cells in a sample.Although the basic approach involved in such detection-based assays isfairly well-established, the existing assays have shortcomings thathinder their wide acceptance. For example, the various reagents thathave been used to lyse bacteria and release their ATP havebroad-specificity; therefore, ATP is released from all susceptiblebacterial and eukaryotic cells present in the sample, which can causefalse-positive readings. In this context, the original DepositedBacteriophage, its progeny, recombinant bacteriophage, or derivativessuch as lytic enzymes will specifically lyse Targeted Bacteria withoutaffecting any other prokaryotic or eukaryotic cells that may be presentin the sample, thus providing means for accurately and specificallyidentifying and detecting Targeted Bacteria.

Epidemiological Typing

The Deposited Bacteriophage, and/or its progeny and derivatives may befurther useful as a tool for the epidemiological typing of TargetedBacteria. For example, one of skill in the art can use the DepositedBacteriophage of the invention to screen a panel of Targeted Bacteriaisolates to aid in the taxonomic identification of the TargetedBacteria, by determining which isolates yield a positive lytic reactionto the Deposited bacteriophage. For example, see (van der Mee-Marquet,N., M. Loessner, et al. (1997). “Evaluation of seven experimental phagesfor inclusion in the international phage set for the epidemiologicaltyping of Listeria monocytogenes.” Appl Environ Microbiol 63(9):3374-3377.).

Preparation of Vaccines or Bacterins

The Deposited Bacteriophage, and/or its progeny and derivatives, alsomay be valuable for preparing bacterial lysates to be used as vaccinesor bacterins. The immunogenicity of such vaccines or bacterins may besuperior to that of so-called dead cell vaccines because phage-mediatedlysis is a more effective and gentler approach for exposing protectiveantigens of bacteria than are approaches used to prepare the lattervaccines. For example, methods commonly used to inactivate bacterialpathogens for dead-cell vaccines, including but not limited to heattreatment, UV-irradiation, and chemical treatment, may deleteriouslyaffect a vaccine's effectiveness by reducing the antigenicity ofrelevant immunological epitopes (Holt, M. E., M. R. Enright, et al.(1990). “Immunisation of pigs with killed cultures of Streptococcus suistype 2.” Res Vet Sci 48(1): 23-27.; Melamed, D., G. Leitner, et al.(1991). “A vaccine against avian colibacillosis based on ultrasonicinactivation of Escherichia coli.” Avian Dis 35(1): 17-22.; Lauvau, G.,S. Vijh, et al. (2001). “Priming of memory but not effector CD8 T cellsby a killed bacterial vaccine.” Science 294(5547): 1735-1739). Thepresence of viable bacteriophage may also serve as an additionalefficacy-enhancing factor, increasing the effectiveness of a phagelysate via their antibacterial effect on the Targeted Bacteria.

The above description of various illustrated embodiments of theinvention is not intended to be exhaustive or to limit the invention tothe precise form disclosed. While specific embodiments of, and examplesfor, the invention are described herein for illustrative purposes,various equivalent modifications are possible within the scope of theinvention, as those skilled in the relevant art will recognize. Theinvention may be practiced in ways other than those particularlydescribed in the foregoing description and examples. The teachingsprovided herein of the invention can be applied to other purposes, otherthan the examples described below.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, manuals, books, or otherdisclosures) in the Background of the Invention, Detailed Description,and Examples is herein incorporated by reference in their entireties.

EXAMPLES Example 1 CPAS-7, CPAS-12, and CPAS-15 Bacteriophage Isolation

The CPAS-7, CPAS-12, and CPAS-15 bacteriophages were isolated fromenvironmental water sources from obtained from agricultural settings orprocessing plants associated with livestock and/or poultry using lysisof distinct strains the Targeted Bacteria to form plaques in bacteriallawns as a means of detecting the presence of bacteriophage having lyticspecificity for the Targeted Bacteria. Plaques were harvested, diluted,and re-plated on bacterial lawns through a process of serial enrichmentuntil a single bacteriophage species, or monophage, was obtained asdetermined by a stable restriction fragment length profile of thebacteriophage DNA. All incubations were performed under anaerobicconditions. The isolates obtained using the technique recited supra maybe cultured using the techniques as set forth herein. The bacteriophagewas deposited with the ATCC.

Example 2 Deposited Bacteriophage Concentration

Concentration of the Deposited bacteriophage may be determined usingtechniques known in the art (Adams, M. H. (1959) Methods of studybacterial viruses. Bacteriophages. London, Interscience Publishers,Ltd.: 443-519.) When a single phage particle encounters a permissivebacterium it will lyse it with the concomitant release of newly formedphage particles. When phages are mixed with host cells and poured in alayer of soft agar on the surface of a nutrient agar plate supportingbacterial growth, the cells will resume growth. In areas where no phagesare present the bacteria will grow to stationary phase, forming a smoothopaque layer or lawn in the overlay. In areas where phages are present,phage progeny from each infected bacterium will infect neighboringbacteria, resulting in a growing zone of lysis full of liberated phagewhich eventually becomes visible to the naked eye as a plaque in theotherwise smooth bacterial lawn. These plaques can be counted, and theirnumber is widely used for expressing phage titer in plaque-forming unitsor PFU. Using this approach, concentration of the Depositedbacteriophage may be determined. Briefly: (1) Various dilutions of theDeposited bacteriophage preparation are prepared; for example, by mixing0.1 ml of phage sample with 9.9 ml of sterile BHI (beef heart infusion)broth. The samples are gently but thoroughly mixed. 0.5 ml of thismixture (which is a 10⁻² dilution of the original sample) is mixed with4.5 ml of sterile BHI broth (10⁻³ dilution). Several 10-fold dilutionsare prepared in a similar fashion; (2) the contents of the tubes (1 mlof various dilutions) are transferred into sterile 10 ml culture tubesand 0.1 ml of host bacterial culture are added. The sample is mixedgently before proceeding immediately to the next step; (3) 3-5 ml ofwarm (45-50° C.) 0.7% BHI agar (top agar) are added. The sample is mixedquickly and very gently. Then, the entire contents of the culture tubeare poured onto a plate containing solidified BHI agar (bottom agar).The plates are slid in circles a few times on the bench top immediatelyafter pouring; (4) after sitting at room temperature for 10 min to allowthe top agar to harden, the plates are inverted and placed into a 37° C.incubator and incubated overnight under anaerobic conditions; (5) thenext morning, the number of plaques on the plate with 30-300 individualwell spaced plaques are counted and the titer calculated and expressedas PFU/ml of the starting sample.

Example 3 Production of the Deposited Bacteriophage

The Deposited bacteriophage is produced using a culture system. Morespecifically, strain of the host Targeted Bacteria or otherclosely-related bacterial species on which the bacteriophage canpropagate is cultured in batch culture, followed by inoculation of thebacteriophage at the pre-determined multiplicity of infection (MOI).Following incubation and bacterial lysis, the bacteriophage is harvestedand purified and/or concentrated to yield phage progeny suitable for theuses enumerated herein. Purification and concentration proceduresincluded variously processing through filtration system(s),centrifugation (including continuous-flow centrifugation) or other wellknown bacteriophage purification and concentration techniques (Adams, M.H. (1959) Methods of study bacterial viruses. Bacteriophages. London,Interscience Publishers, Ltd.: 443-519.)

The invention provides compositions comprising active viral particles ofthe bacteriophage capable of lysing strains of Targeted Bacteria. Theconcentration of bacteriophage is determined using phage titrationprotocols. The final concentration of the bacteriophage is adjusted byconcentration, if a more concentrated phage composition is desired, viafiltration, centrifugation, or other means, or by dilution, if a lessconcentrated phage composition is desired, with water or buffer to yielda phage titer of 10⁶ to 10¹² PFU/ml, preferably 10¹⁰ to 10¹¹ PFU/ml. Theresulting bacteriophage compositions are generally stored at 4° C.;alternatively, preparations can be freeze or spray-dried for storage, orcan be encapsulated and stabilized with protein, lipid, polysaccharide,or mixtures thereof. Upon reconstitution, the phage titer can beverified using phage titration protocols and host bacteria. One of skillin the art is capable of determining bacteriophage titers using widelyknown bacteriophage assay techniques (Adams, M. H. (1959) Methods ofstudy bacterial viruses. Bacteriophages. London, IntersciencePublishers, Ltd.: 443-519.)

Example 4 Application of the Deposited Bacteriophage for Preservation ofFood Products

The bacteriophage produced using the methods of the present inventionmay be dispersed in an appropriate aqueous solution or lyophilized orfreeze-dried powder and applied to the surface of food products.Alternatively, the bacteriophage may be included with a cheese cultureor other microbially active foodstuff prior to or during processing.

Example 5 Isolation of the Bacteriophage DNA

Bacteriophage DNA, a derivative of the bacteriophage, can be used forvarious applications such as for preparing DNA-based vaccines, and alsofor analytical purposes, for identifying the bacteriophage such as RFLPprofile determination and comparison. Phage DNA can be isolated using asuitable commercial kit such as the Lambda Mini Kit (Qiagen, Inc.;Valencia, Calif.) or the standard phenol extraction technique. Forexample, 0.75 ml of phage in phosphate-buffered saline solution at atiter of 10⁸-10¹¹ PFU/ml is collected. 10 μl of Proteinase K (20 mg/ml)and 2 μl of RNAse (10 mg/ml) is added, followed by incubation at 37° C.for 30 minutes, and a subsequent incubation at 56° C. for 30 minutes.Following incubation, 75 μl of a mixture of 10% SDS (0.1 ml), 0.5 M EDTA(0.1 ml) and 0.8 ml of water is added and incubated at room temperaturefor 5 min. 0.75 ml of a phenol:chloroform:isoamylalcohol (25:24:1)solution is mixed well with the sample, followed by centrifugation at13,000 RPM for five (5) min. Next, the supernatant (approximately 600μl) is carefully removed and transferred to a clean eppendorf tube. 0.6ml of chloroform is added to the supernatant, mixed well, andcentrifuged at 13,000 RPM for five (5) min. The supernatant is thencarefully extracted (approximately 500 μl). Next, 0.1 volumes of 3Msodium acetate (40 ml) is added to the solution, followed by 2.5 volumesof cold 95% ethanol (1 ml) to precipitate the bacteriophage DNA. Thesolution is allowed to incubate at −20° C. for 1 hour, followed bycentrifugation at 13,000 RPM for thirty (30) min. Followingcentrifugation, the pellet is washed with 1 ml of 70% cold ethanol, andthe supernatant is poured from the pellet. The pellet is allowed to airdry, and is then resuspended in 30-300 μl of TE (10 mM tris-HCL,pH=8.0-8.5, 1 mM EDTA).

Example 6 Restriction Fragment Length Polymorphism (RFLP) Profile

RFLP can be used to identify the Deposited bacteriophage or its progeny.The progeny will have a substantially equivalent RFLP DNA profile as theRFLP DNA profile of the original bacteriophage. A reference RFLP profileof the Deposited bacteriophage is shown in FIG. 1. DNA was isolated fromthe bacteriophage using Qiagen Plasmid Miniprep or Midiprep kits(Valencia, Calif.) according to the manufacturer's directions. The DNAwas quantitated by measuring absorbance at 260 nm. Approximately 0.5-1μg of DNA was digested with an appropriate restriction enzyme (FIG. 1),and RFLP profile was determined on 1% agarose gel after staining withethidium bromide.

Example 7 Lytic Specificity of the Bacteriophage

Forty-six Clostridium perfringens strains were screened for theirsusceptibility to the bacteriophage by the drop-on-lawn method, alsoknown as the “spot test” method. Strains were streaked onto BHI agarplates and incubated at 37° C. overnight anaerobically. One colony ofeach strain was inoculated into a separate well of a 96-well microtiterplate containing BHI broth and incubated at 37° C. until the OD600reached 0.2-0.3. One hundred microliters of each strain were mixed withBHI soft agar and poured onto a BHI agar plate. After the soft agarhardened 10 μl of the bacteriophage were spotted in triplicate onto theplates inoculated with the strains of Targeted Bacteria. Lytic activitywas observed after overnight anaerobic incubation at 37° C. Lyticspecificity results are presented in Table 1. At least one of thedeposited bacteriophages lysed 41 (89%) of the 46 strains of TargetedBacteria examined. In contrast, none (0.0%) of 40 non-Clostridiumperfringens strains of other bacterial species (Table 2) were lysed.

Example 8 Detection of Targeted Bacteria in Food Samples

The bacteriophage or its derivative, such as lytic enzyme, producedusing the methods of the present invention is used to specifically lyseTargeted Bacteria without affecting any other prokaryotic or eukaryoticcells that may be present in the sample; thus, specifically elicitingtheir release of measurable bacterial products such as AK or ATP.Briefly: (1) Samples of the food to be analyzed are obtained andsuspended in appropriate buffer, (2) The Deposited bacteriophage isadded to the suspensions, as a result of which the Targeted Bacteriacells present in the samples are lysed and their ATP is released, (3) Aluciferin+luciferase preparation is added to the mixtures, and (5) Themixtures' luminescence is measured within 60 sec, and the results aredisplayed on a handheld luminometer. It may be possible to establish acorrelation between the luminometer readings and the number of TargetedBacteria cells lysed (in general, the average amount of ATP perbacterial cell is 0.5-1.0 fg; precise correlation between theluminometer readings and the number of Targeted Bacteria cells should beexperimentally established). If Targeted Bacteria cells are not presentin the food samples analyzed, bacterial lysis and ATP-release will notoccur.

Example 9 Preparing Vaccines and Bacterins

One example of utilizing bacteriophages to prepare vaccines andbacterins is to use the lytic Deposited bacteriophage to lyse specificstrains of the Targeted Bacteria, which will yield bacterial lysatescontaining minimally-affected immunological epitopes of the bacteria.The phage may be removed from the final vaccine/bacterin preparation.Alternatively, it may be retained unaltered in the preparation becauseits lytic activity against Targeted Bacteria that may be present in themammalian organism being vaccinated may increase the preparation'sefficacy. In one preferred embodiment of the present invention: (i) themost prevalent, problematic strains of the Targeted Bacteria are chosenso that the vaccine/bacterin contains the immunological epitopes thatare most relevant for protecting against the infection, and (ii) thebacteriophage is kept unaltered in the final vaccine/bacterin, at levelsranging from 10⁶-10¹⁰ PFU/ml.

Bacteriophage-based vaccines and bacterins also may be prepared by usingderivatives of the Deposited bacteriophage to lyse the TargetedBacteria. An example of the general methodology can be briefly outlinedfrom a recent study (Panthel, K., W. Jechlinger, et al. (2003).“Helicobacter pylori ghosts by PhiX protein E-mediated inactivation andtheir evaluation as vaccine candidates.” Infect Immun 71(1): 109-16.) ofan Helicobacter pylori bacterin. The authors used Clostridiumperfringens-H. pylori shuttle plasmid pHe12 and lysis gene e ofbacteriophage φX174 to construct H. pylori lysis plasmid pHPC38, whichthey introduced into H. pylori strain P79. At a pre-determined time, theauthors triggered e gene-expression in order to elicit bacterial lysis,and they found that the lysate protected BALB/c mice against H. pyloriinfection.

1. An isolated bacteriophage strain CPAS-7, CPAS-12, or CPAS-15deposited under ATCC accession number PTA-8478, PTA-8479 or PTA-8480respectively, having lytic activity against Clostridium perfringensstrains, and variants thereof, wherein said variants have the samephenotypic characteristics of said bacteriophage strain and the samelytic activity of said bacteriophage strain against Clostridiumperfringens strains.
 2. An isolated progeny of the bacteriophage strainof claim 1, wherein said progeny has the same phenotypic characteristicsof said bacteriophage strain and the same lytic activity of saidbacteriophage strain against Clostridium perfringens strains.
 3. Acomposition comprising the isolated bacteriophage strain of claim
 1. 4.A composition comprising the isolated bacteriophage strain of claim 2.